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International Journal of Bioprinting Expanding 3D cell proliferation with DLP bioprinting

a total light exposure time of 36.13 s (31.13 s plus an was performed for each time course using a Live/Dead
®
additional 5 s). cell viability kit containing calcein-AM and ethidium
After printing, the fabricated scaffold was removed homodimer-1. The printed hydrogel was prepared in
from the plate and incubated in high-glucose DMEM a cylindrical shape with dimensions of 8 mm × 1 mm
supplemented with 10% FBS, 1% L-glutamine, and 1% P/S. (width and height, respectively). During the time course
experiment (1, 3, 5, 7, 14, 21, 28, and 35 days), 500 µL of
2.2.4. Photorheological analysis staining solution was added to the sample following the
To evaluate the rheological characteristics of the produced manufacturer’s protocol, and the sample was incubated at
F-GelMA bioink during the crosslinking process, we 37°C for 15 min. The cells were seeded at a concentration
performed a photorheological experiment using a HAKKE™ of 2 × 10 cells/mL. Cell viability was calculated as the
6
MARS™ 40 (Thermo Fisher Scientific, Waltham, MA, USA) proportion of live cells relative to the total number of cells,
equipped with a light module accessory. Measurements as determined by image analysis using a Lionheart™ FX
were taken with a 100 μm gap between the glass and the microscope (BioTek Instruments, Winooski, VT, USA).
bottom to load the bioink. The light intensity is set to
2.45 mW/cm . We used the OmniCure LX 505 (Lumen 2.6. Fabrication and accuracy analysis of three
2
®
Dynamics; Mississauga, ON, Canada) as the light source, different sizes of microchannel in a DLP-printed 3D
equipped with a 405 nm light-emitting diode channel hydrogel scaffold
that matched the wavelength of the DLP printer (IM2). Three different sizes of cross-sectioned microchannel
We conducted an oscillation experiment at 37°C with an hydrogels were fabricated, with dimensions of 3 mm × 3
oscillatory shear strain of 0.008% and a frequency of 20 Hz. mm × 3 mm (width, length, and height, respectively) for
immunocytochemical analysis. To prepare the samples
2.3. Scanning electron microscopy for measurement, they were placed on a microscope slide
To confirm the morphological characterization of the (Paul Marienfeld, Lauda-Königshofen, Germany), and
printed F-GelMA via DLP printing, we measured the cross- internal sections were cut using 22 mm × 22 mm cover
section of vertically cut 3D-printed scaffolds using a field- glass (Fisherfinest Premium cover glass, 12548B, Fisher
®
emission scanning electron microscope (FE-SEM; SU8010, Scientific, OH, USA). In this study, after a 3-day cell
Hitachi, Tokyo, Japan). The scaffold had a rectangular shape, stabilization period post-printing, a media flow environment
with dimensions of 6 mm × 6 mm × 6 mm (width, length, of 30 rpm orbital shaking was introduced to a non-shaking
and height, respectively). It was printed using F-GelMA culture. Observation and analysis were conducted through
bioink and deionized water without cells. The printed α-tubulin/DAPI immunocytochemistry staining starting
F-GelMA hydrogels were first observed under a light from 5 days at different time course (5, 7, 14, 21, 28, and
microscope (Nikon ECLIPSE Ts2; Nikon, Tokyo, Japan) at 35 days). The cross-sectioned samples were fixed with 4%
4× and 10× magnifications. Subsequently, the surface of the paraformaldehyde at 37°C for 10 min and permeabilized
scaffold was quickly blotted and frozen in liquid nitrogen with 0.25% Triton X-100 in PBS for 10 min. After rinsing
for 10 min. After lyophilization (3 days in a freeze-dryer in PBS, the samples were blocked with 5% bovine serum
at -80°C), the frozen hydrogel was vertically cut and fixed albumin and 0.1% Triton X-100 in PBS at 37°C for 1
onto a stub using carbon tape. The sample was then coated h. Primary antibody was administered for 1 h at 37°C,
with platinum for 2 min. We measured the pores size of using mouse monoclonal anti-α-tubulin antibody 12G10
the scaffolds using SEM images (300× magnification) and (dilution 1:300; Developmental Studies Hybridoma Bank,
analyzed the data using ImageJ 1.52v software (National Iowa City, MA, USA). After rinsing five times in PBS, a
Institutes of Health, Bethesda, Maryland, USA). secondary antibody was administered for a 1-h incubation

2.4. Cell culture at 37°C using goat anti-mouse IgG H&L (dilution 1:300;
Bovine ear fibroblast cells (BEFCs), generously provided Invitrogen, Carlsbad, CA, USA). The nuclei were stained
by LARTbio, Seoul, South Korea, were cultured and with 4’,6-diamidino-2-phenylindole dihydrochloride
maintained at 37°C and 5% CO in high-glucose DMEM (DAPI) (dilution 1:1000; 10236276001, Sigma Aldrich,
2
supplemented with 10% (v/v) FBS, 1% (v/v) L-glutamine, St. Louis, MO, USA), and after a 15 min incubation at
and 1% (v/v) P/S. The cells were passaged twice using 37°C, the samples were washed in PBS. Fluorescent signals
0.05% trypsin-EDTA for 7 days, and the media were were observed using a Lionheart™ FX microscope (BioTek
changed every 2 days. Instruments, Winooski, VT, USA). The analysis for
α-tubulin confluency was measured as a percentage using
2.5. Live/Dead assay ImageJ 1.25v software (U.S. National Institutes of Health,
To evaluate cell viability (on two-dimensional [2D] cell Bethesda, Maryland, USA). The confluency of α-tubulin
culture plate and the surface of printed hydrogel), staining (Equation I) was analyzed based on the difference between

Volume 10 Issue 3 (2024) 411 doi: 10.36922/ijb.2219